The present invention relates to recombinant host cells that comprise a heterologous, polypeptide-encoding polynucleotide segment which is stably integrated into a chromosome and which is under control of an endogenous promoter. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomanas mobilis, recombinant Escherichia coli and other enterobacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. This invention also relates to mutations that enhance production of proteins encoded by chromosomally-integrated, heterologous genes which are expressed under the control of an endogenous promoter, and to methods of identifying such mutations.
During glycolysis, cells convert simple sugars, such as glucose, into pyruvic acid, with a net production of ATP and NADH. In the absence of a functioning electron transport system for oxidative phosphorylation, at least 95% of the pyruvic acid is consumed in short pathways which regenerate NAD+, an obligate requirement for continued glycolysis and ATP production. The waste products of these NAD+ regeneration systems are commonly referred to as fermentation products.
Microorganisms are particularly diverse in the array of fermentation products which are specific for each genus. See, for example, Krieg, N. R., and J. G. Holt, eds. [1984] BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY (Williams & Wilkins Co., Baltimore). These products include organic acids, such as lactate, acetate, succinate, and butyrate, as well as neutral products like ethanol, butanol, acetone, and butanediol. Indeed, the diversity of fermentation products from bacteria has led to their use as a primary determinant in taxonomy. Krieg and Holt [1984], supra.
End products of fermentation share several fundamental features. They are relatively nontoxic under the conditions in which they are initially produced but become more toxic upon accumulation. They are more reduced than pyruvate because their immediate precursors have served as terminal electron acceptors during glycolysis. The microbial production of these fermentation products forms the basis for our traditional and most economically successful applications of biotechnology and includes dairy products, meats, beverages, and fuels.
Most fuel ethanol is currently produced from hexose sugars in corn starch or cane syrup utilizing either Saccharomyces cerevisiae or Zymomanas mobilis (Z. mobilis). However, these are relatively expensive sources of biomass sugars and have competing value as foods. In addition, during fermentation much of the hexose is necessarily converted back to biomass, comprising microbial cells, rather than to ethanol. For conventional ethanol-producing microorganisms, this biomass has limited commercial value at best, for instance, as a nutritional supplement, and therefore, represents inefficient utilization of the expensive sugar substrate.
Starches and hexose sugars represent only a fraction of the total carbohydrates in plants. The dominant forms of plant carbohydrate in stems, leaves, hulls, husks, cobs, etc., are the structural wall polymers, cellulose and hemicellulose. Hydrolysis of these polymers releases a mixture of neutral sugars which include glucose, xylose, mannose, galactose, and arabinose. No known organism in nature can rapidly and efficiently metabolize all of these sugars, particularly the pentoses, into ethanol or any other single product of value.
Escherichia coli (E. coli) and related enteric bacteria are the main commercially useful microorganisms that are capable of metabolizing the entire range of biomass-derived sugars by fermentation under anaerobic conditions. However, under anaerobic fermentation conditions, these organisms convert sugars to a mixture of soluble products, including small amounts of ethanol, that cannot be separated economically. See Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J. F. Preston [1987] Appl. Environ. Microbiol. 53: 2420-2425. Thus, such enteric bacteria efficiently utilize the entire range of biomass-derived sugars but fail to produce a product of sufficient yield and uniformity to be commercially valuable.
Accordingly, there is a need for microorganisms which combine the efficient metabolism of the entire range of biomass-derived sugars, which is exhibited by certain enteric bacteria, such as E. coli, with the ability to produce high levels of a single, predominant, soluble fermentation product of commercial value, such as ethanol. Further, there is a continuing need for such organisms that can produce microbial biomass comprising additional products, such as commercially valuable proteins, in sufficient yield and quality for economical recovery.
Fermentation pathways transform pyruvic acid into a mixture of acidic and neutral products. Two pathways dominate in enteric bacteria such as E. coli. Lactate dehydrogenase catalyzes the reduction of pyruvate to lactic acid, directly oxidizing NADH to NAD+. The second pathway, involving pyruvate formate-lyase, is more complicated. Pyruvate formate-lyase, which catalyzes the cleavage of pyruvate to formate plus acetyl-coenzyme A, is a central enzyme of the anaerobic metabolism of E. coli, because under anaerobiosis this enzyme is responsible for metabolizing a large fraction of pyruvate. The E. coli gene encoding pyruvate formatelyase (pfl gene) has been cloned and sequenced. See Christiansen, L., and S. Pedersen (1981) Mol. Gen. Genet. 181: 548-551; Rodel, W., W. Plaga, R. Frank, and J. Knappe [1988] Eur. J. Biochem. 177: 153-158. The pfl gene is preceded by multiple promoters, and it is induced to high levels of expression by anaerobiosis. See Sawers, G., and A. Bock [1988] J. Bacteriol. 170: 5330-5336.
The DNA used to provide ethanol-production genes for a recombinant host of the subject invention is isolated, for example, from Z. mobilis. This is a microorganism with unusual metabolic characteristics which is commonly found in plant saps and in honey. Wild-type Z. mobilis has long served as a natural inoculum for the fermentation of the Agave sap to produce pulque, a Mexican alcoholic beverage, and as an inoculum for palm wines. As noted above, this organism is also used for fuel ethanol production and has been reported to be capable of ethanol production rates which are substantially higher than those of yeasts.
Although Z. mobilis is nutritionally simple and capable of synthesizing amino acids, nucleotides and vitamins, the range of sugars metabolized by this organism is very limited and normally consists of glucose, fructose and sucrose. Substrate level phosphorylation from the fermentation of these sugars is the sole source of energy for biosynthesis and homeostasis. Z. mobilis is incapable of growth without a fermentable sugar even in rich medium such as nutrient broth.
In Z. mobilis, two enzymes, pyruvate decarboxylase (PDC) and alcohol dehydrogenase, particularly form II (ADHII), are required to convert pyruvate to ethanol and regenerate NAD+. High levels of the individual proteins are found in the cytoplasm of Z. mobilis, ranging from 2% to 5% each of the soluble protein. Such high levels are presumed to be essential for the high rates of NADH oxidation and glycolytic flux required for energy production. The cloning and sequencing of Z. mobilis pdc and adhB genes encoding PDC and ADHII, has been previously reported. See Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffman, and L. O. Ingram [1987] J. Bacteriol. 169: 949-954; Conway, T., G. W. Sewell, Y. A. Osman, and L. O. Ingram [1987] J. Bacteriol. 169: 2591-2597; Brau, B., and H. Sahm [1986] Arch. Microbiol. 146:105-110; Brau, B., and H. Sahm [1986] Arch. Microbiol. 144: 296-301; Neale, A. D., R. K. Scopes, R. E. H. Wettenhall, and N. J. Hoogenraad [1987] Nucleic Acid. Res. 15: 1753-1761; Ingram, L. O., and T. Conway [1988] Appl. Environ. Microbiol. 54: 397-404; Ingram, L. O, T. Conway, D. P. Clark, G. W. Sewell, and J. F. Preston [1987] Appl. Environ. Microbiol. 53: 2420-2425.
Molecular genetics offers the potential to combine in a single organism the pathway for anaerobic metabolism in pentose-utilizing enteric bacteria, such as E. coli, and the efficient pathway for ethanol production from an ethanol producer such as Z. mobilis. Thus, expression of the Z. mobilis pdc gene in enteric bacteria such as E. coli, Ervinia chrysanthemi and Klebsiella planticola partially diverts the flow of pyruvate to ethanol as a fermentation product by using low levels of native ADH activity. More efficient ethanol production and higher concentrations of ethanol have been obtained with recombinant E. coli harboring the Z. mobilis genes encoding PDC and ADHII on a multi-copy plasmid. See Ingram et al. [1987], supra; Neale, A. D., R. K. Scopes, and J. M. Kelly [1988] Appl. Microbiol. Biotechnol. 29: 162-167. E. coli B (pLOI297) and E. coli ATCC 15224 (pLOI297) strains are superior constructs in terms of ethanol production and environmental hardiness. See Alterthum, F., and L. O. Ingram [1989] Appl. Environ. Microbiol. 55: 1943-1948. These recombinant E. coli efficiently ferment glucose, lactose, and xylose to ethanol.
The recombinant E. coli described above achieved useful levels of ethanol production using plasmid-borne ethanol-production genes from Z. mobilis. Further, initial testing of ethanol production in prototype strains was facilitated by placing the exogenous genes on a multi-copy plasmid. However, the exogenous genes were not completely stable because of the inherent instability of plasmids in the absence of selective pressure to ensure their retention in the host cell. Due to plasmid incompatibilities, moreover, the use of a typical E. coli expression plasmid for the ethanol-production genes precludes the most convenient means for introduction into a basic commercial ethanol- producer strain of additional exogenous genes for production of other selected products, such as valuable proteins.